Alternative Lactose Catabolic Pathway in Lactococcus lactis IL1403

Abstract

In this study, we present a glimpse of the diversity of Lactococcus lactis subsp. lactis IL1403 ␤-galactosidase phenotype-negative mutants isolated by negative selection on solid media containing cellobiose or lactose and X-Gal (5-bromo-4-chloro-3-indolyl-␤-D-galactopyranoside), and we identify several genes essential for lactose assimilation. Among these are ccpA (encoding catabolite control protein A), bglS (encoding phospho-␤-glucosidase), and several genes from the Leloir pathway gene cluster encoding proteins presumably essential for lactose metabolism. The functions of these genes were demonstrated by their disruption and testing of the growth of resultant mutants in lactose-containing media. By examining the ccpA and bglS mutants for phospho-␤-galactosidase activity, we showed that expression of bglS is not under strong control of CcpA. Moreover, this analysis revealed that although BglS is homologous to a putative phospho-␤-glucosidase, it also exhibits phospho-␤-galactosidase activity and is the major enzyme in L. lactis IL1403 involved in lactose hydrolysis.Bacteria have evolved three different systems for the assimilation of the main milk sugar, lactose, which differ in their phosphorylation states, intermediate metabolites, and bioenergetics. They include the group translocation systems (13, 39) and the primary (15) and secondary (27, 37) transport systems.During transport by the bioenergetically most efficient group translocation system, the lactose-specific phosphoenolpyruvate (PEP)-dependent phosphotransferase system (PTS) (lac-PTS), lactose is phosphorylated at the C-6 carbon, and the internalized lactose 6-phosphate is degraded into galactose 6-phosphate and glucose by phospho-␤-galactosidase. It has been suggested that in some cases lactose 6-phosphate can be hydrolyzed by ␤-glycosidases specific for ␤-glucoside sugars, that is, by P-␤-glucosidases (46). This seems to be supported by sequence similarities between P-␤-galactosidase and P-␤-glucosidase enzymes, both of which, according to the nomenclature of Henrissat (24), belong to family I of glycohydrolases.In primary and secondary transport systems lactose is not phosphorylated, and after internalization, it is hydrolyzed by ␤-galactosidase, yielding glucose and galactose. Galactose is subsequently metabolized through the Leloir pathway. Uptake of lactose via primary transport systems depends on hydrolysis of ATP, which provides energy for translocation of the substrate by an ATPase. Secondary transport systems use the energy from solute gradients, and in sugar translocation different types of mechanisms are involved, such as symport, antiport, and uniport.Lactococcus lactis is a lactic acid bacterium (LAB) used in the dairy industry as a starter culture. Some strains of this species are able to ferment lactose present in milk very rapidly.In these strains lactose is transported by the lac-PTS and hydrolyzed by P-␤-galactosidase. The galactose 6-phosphate formed is further metabolized via the tagatose 6-phosphate pathway. In lactococci, oper...

Lactose and β-Glucosides Metabolism and Its Regulation in Lactococcus lactis: A Review

“…2.A.2.2.1) with a proven high lactose-transport rate. The lack of LacS involvement in lactose transport is confirmed by the fact that disruption of lacS in L. lactis IL1403 had a minor effect on lactose assimilation (Aleksandrzak-Piekarczyk et al, 2005). Another indispensable factor in lactose assimilation, the -galactosidase enzyme, is also encoded by the genomes of L. lactis IL1403 and NCDO2054 strains.…”

“…Another indispensable factor in lactose assimilation, the -galactosidase enzyme, is also encoded by the genomes of L. lactis IL1403 and NCDO2054 strains. In spite of the high similarity in the protein level of both enzymes, -galactosidase of L. lactis NCDO2054, in contrast to the one of L. lactis IL1403 (Aleksandrzak-Piekarczyk et al, 2005), seems to be highly active and strongly regulated (Griffin et al, 1996). It has been suggested that the lacZ gene of L. lactis IL1403 may not be expressed or the encoded enzyme may be inactive since this strain does not exhibit -galactosidase activity (AleksandrzakPiekarczyk et al, 2005).…”

“…Until recently (Aleksandrzak et al, 2000;Aleksandrzak-Piekarczyk et al, 2005Kowalczyk et al, 2008), little information on the organization in L. lactis strains of chromosomal alternative lactose utilization genes has been available. It was shown that in lac-plasmid-free, and thus lactose-negative L. lactis IL1403, the ability to assimilate lactose can be induced in two ways: (i) by the presence of cellobiose or (ii) by inactivation of CcpA (Aleksandrzak et al, 2000;Aleksandrzak-Piekarczyk et al, 2005).…”

“…Among them, catabolite repression (Aleksandrzak-Piekarczyk et al, 2005 and transcriptional antitermination through the BglR protein (Bardowski et al, 1994) were shown to be operational in L. lactis. The antitermination mechanism allows for expression of -glucoside-specific genes in the absence of a metabolically preferred carbon source, such as glucose (Rutberg, 1997).…”

“…However, the data concerning their involvement in -glucosides assimilation are rather scarce in scientific literature. It has only been demonstrated that a P--glucosidase, BglS, is responsible for hydrolysis of cellobiose, but not of salicin in L. lactis IL1403 (Aleksandrzak-Piekarczyk et al, 2005) (Fig. 1).…”